The present invention concerns hepatitis C virus (hereinafter called HCV) oligonucleotides and a system to determine HCV genotypes with polymerase chain reaction (PCR) utilizing those oligonucleotides as primers.
Viral hepatitis of which DNA and RNA of the causative viruses have been elucidated, and their diagnosis and even prevention in some cases have been established, are hepatitis A and hepatitis B. The general name NANB (non-A, non-B) hepatitis was given to the other forms of viral hepatitis.
Post-transfusion hepatitis was remarkably reduced after introduction of diagnostic systems for screening hepatitis B virus in transfusion bloods. However, there are still an estimated 280,000 annual cases of post-transfusion hepatitis caused by NANB hepatitis virus in Japan.
NANB hepatitis viruses were recently named C, D and E according to their types, and scientists started a world wide effort to conduct research for the causative viruses.
In 1988, Chiron Corp. claimed that it had succeeded in cloning RNA virus genome, which it termed hepatitis C virus (HCV), as the causative agent of NANB hepatitis and reported on its nucleotide sequence (British Patent 2,212,511 which is the equivalent of European Patent Application 0,318,216). HCV (c100-3) antibody detection systems based on the sequence are now being introduced for screening of transfusion bloods and for diagnosis of patients in Japan and in many other countries.
The detection systems for the c100-3 antibody have proven their partial association with NANB hepatitis; however, they capture only about 70% of carriers and chronic hepatitis patients, or they fail to detect the antibody in acute phase infection, thus leaving problems yet to be solved even after development of the c100-3 antibody by Chiron Corp.
The genome structure of HCV, or NANB hepatitis having relation to c100-3 antibody, has single stranded RNA which suggests some relationship to Flaviviruses and Pestiviruses. From the comparison of those genome structures, regions coding core protein (C), envelope protein (E) and non-structure protein (NS) were found in HCV. c100-3 antibody detected by Chiron's ELISA kit is thought to recognize a part of the border region of NS3/NS4.
The course of NANB hepatitis is troublesome and many cases of horizontal transmission are considered to become carriers and then to develop chronic hepatitis. In addition, most patients with chronic hepatitis develop liver cirrhosis, then hepatocellular carcinoma. It is therefore very imperative to isolate the virus itself and to develop effective diagnostic reagents enabling earlier diagnosis.
The presence of a number of NANB hepatitis which can not be diagnosed by Chiron's HCV (c100-3) antibody detection kits suggests the importance of detection of antibody to core protein instead of antibody to non-structural protein. From that view point, the genome of the core region and the protein coded for by this region have been studied world wide.
From the two-by-two comparison of nucleotide sequences of various HCV isolates, homologies for the core region, which is rather conservative, were found to be less than 90% in some cases. In addition, more variability was found in the envelope region (which is thought to be important for development of vaccine); less than 60% homology was found between some isolates. The sequence diversity between strains reflects the features of HCV as RNA virus.
A rapid and simple method of classifying of HCV genomes into groups, therefore, would be very useful for diagnosis, therapy and prevention of NANB hepatitis.
Determination of HCV genotypes could be useful in the following medical and social applications:
(1) identification of infectious sources in vertical (mother-to-baby) transmission and horizontal transmission;
(2) clarification of genotype-specific features in the course, condition and prediction of recuperation of NANB liver diseases and making a guide of therapy therefore:
(3) increasing the detection sensitivity of HCV by nucleic acid detection system or antibody detection system based on the study of diversity in nucleotide or amino acid sequences among HCV isolates of distinct genotypes;
(4) production of type-specific immunoglobulin (IgG) and vaccine based on the study of type-specific conservative regions in the envelope gene of various genotypes; and
(5) rapid and effective prevention by selecting an appropriate IgG or vaccine for each genotype.
The inventors have obtained various HCV-RNAs from sera of humans and chimpanzees, and cloned cDNAs of HCV genomes in non-coding regions and coding regions for core and envelope proteins, followed by determination of nucleotide sequences (Japanese Patent Applications 196175/91, 287402/91 and 360441/91), and sought to find a method to determine HCV genotypes and oligonucleotide primers to be used in said method, which would be useful in diagnosis, therapy, prevention, identification of infectious source, and epidemiological study on HCV. As a result, variation of nucleotide sequence characteristics in some groups in the core region, which was thought to be rather conservative, was found, and much more variation was found in the envelope region.